The long-term objective of this proposal is to elucidate the mechanisms by which cell type-specific genes are activated during embryo development. The fundamental question of how individual cell types arise from the fertilized egg remains largely unanswered in deuterostome organisms. Sea urchin embryos will be used as a model system to address the key issues of cell type-specific gene activation during development Sea urchins offer unique opportunities for developmental analysis because of their close evolutionary and developmental relationship to vertebrates. The proposed experiments will investigate a well-described sea urchin cell type, the aboral ectoderm, by taking advantage of two discoveries made in the past granting period. First, a Wnt-beta-catenin- Tcf/Lef signal transduction pathway has been show to be essential for the polarization of primitive ectoderm into definitive oral and aboral ectoderm territories. Second, the cis-regulatory elements within the aboral ectoderm-specific Spec2a enhancer responsible for spatially regulated transcription of the Spec2a gene have been identified. Together these findings provide the opportunity to link the cell specification events mediated by the Wnt-beta-catenin-Tcf/Lef pathway with the regional activation of transcription factors required for aboral ectoderm-specific gene expression. The hypothesis that the transcriptional activation of aboral ectoderm repressors is linked to Wnt-beta-catenin-Tcf/Lef signaling will be tested. The specific aims are to: (1) define the properties of the cis-regulatory elements and DNA-binding proteins within the Spec2a enhancer that confer aboral ectoderm specificity; (2) Test whether SpKrox1, a vegetally-localized transcription factor, functions as a transcriptional repressor in vegetal cell lineages; (3) Determine the signaling pathway leading to ectoderm polarization and aboral ectoderm- specific gene expression; and (4) Monitor the changes in cis-regulatory elements within the Spec2a enhancer during sea urchin evolution. By focusing on the sea urchin embryo, this proposal will provide important mechanistic information on cell type-specific gene activation that cannot be obtained using any other nonvertebrate deuterostome.